Abstract This proposal challenges the current dogma that `non-contact' anterior cruciate ligament (ACL) ruptures are due to sudden awkward landing or overload event. In the past funding cycle we developed the first evidence that the ACL can also fail under repetitive sub-maximal loading (Lipps 2013), helping to explain why an ACL can fail during a routine athletic maneuver. Pilot data suggest that this fatigue failure is associated with an accumulation of microdamage near the proximal femoral enthesis. We request funds to test this hypothesis. In AIM 1 60 pairs of knees from sex- and size-matched younger cadavers (30 males, 30 females) will be positioned at an initial flexion angle of 15o via pretensioned knee muscle-tendon equivalents that allow the knee to flex under a distal impulsive test load. ACL fatigue life will be determined via repetitive sub-maximal four-times bodyweight impulsive 3-D compound loading (compression + trans knee muscle loading + flexion moment + knee abduction moment + internal tibial axial torque) applied to each knee. A Cox regression model will be used to rank the relative importance of sex, internal femoral axial rotation range of motion (ROM) and lateral tibial slope in limiting ACL fatigue life. In AIM 2a we will use histological images and a binomial exact test to determine the percentage of the knees undergoing AIM 1 fatigue testing that exhibit microdamage at or near the femoral enthesis exceeding the +3*SD area found in the 32 untested, paired, control knees. Similarly, in AIM 2b we will salvage ACL femoral enthesis explants in 60 healthy young adults (30 females) aged 14-25 years undergoing routine reconstruction of their ACL and determine the percentage of explants exhibiting microdamage area exceeding the +3*SD value measured in 30 untested age- and sex-matched cadaveric controls. If greater-than-normal ACL microdamage is found in fatigue-tested knees in vitro (AIM 2a) and in ACL-injured patients in vivo (AIM 2b) this suggests the ACL can fail due to an accumulation of fatigue damage. This would imply that there may be time to modify athlete work-rest cycles to reduce ACL injury risk by arresting the injury cycle (Olsen 2006) and allowing the ACL to heal (Maekawa 1996).